Rapid identification of Candida isolates to the species level in the clinical laboratory has become more important because the incidence of candidiasis continues to rise in proportion to a growing number of patients at risk for infection with Candida albicans and, recently, with innately azole-resistant non-albicans Candida species (Fridkin and Jarvis, Clin. Microbiol. Rev., 9:499-511, 1996; Jarvis, Clin. Infect. Dis., 20:1526-1530, 1995; Wingard, Clin. Infect. Dis., 20:115-125, 1995). This patient population has increased as a result of more intensive regimens of cancer therapy, complications of abdominal or cardiothoracic surgery, organ transplantations, burns, and trauma. Affected patients may be immunocompromised or not, and common risk factors include prolonged broad-spectrum antibiotic therapy, invasive devices such as indwelling Hickman catheters, and/or prolonged hospital stays (Fridkin and Jarvis, Clin. Miciobiol. Rev., 9:499-511, 1996; Jarvis, Clin. Infect. Dis., 20:1526-1530, 1995; Wenzel, Clin. Infect. Dis., 20:1531-1534, 1995). Under these conditions, an antibiotic-resistant replacement flora, including Candida species, can proliferate in the gut and invade deep tissues from mucosal foci. This is especially true when mucosal integrity has been disrupted as a result of chemotherapy or surgery. In addition, as the number of risk factors increases, the odds of developing candidiasis multiply (Wenzel, Clin. Infect. Dis., 20:1531-1534, 1995). Consequently, rapid identification to the species level is necessary for more timely, targeted, and effective antifungal therapy and to facilitate hospital infection control measures.
Identification of Candida species by conventional morphology and assimilation tests can be time consuming and laborious, especially for difficult-to-grow or morphologically atypical species (Warren and Hazen, “Cryptococcus and other yeasts of medical importance,” In: Manual of Clinical Microbiology, 6th Edition, ed. by Murray et al., Washington, D.C.: American Society for Microbiology, 1995, pp. 723-737). Therefore, reliable, reproducible tests for the rapid identification of Candida isolates to the species level would be clinically and epidemiologically important.
Molecular methods for identification of Candida sp. have been developed. Some methods of molecular identification of fungi also can be very difficult or cumbersome to perform or require expensive, specialized equipment (e.g., Sandhu et al., J. Clin. Microbiol., 35:1894-1896, 1997; Sandhu et al., J. Clin. Microbiol., 33:2913-2919, 1995; Turenne et al., J. Clin. Microbiol., 37:1846-1851, 1999). For instance, DNA sequencing, determination of sequence length polymorphisms, single-stranded conformational polymorphism analysis and restriction fragment length polymorphism analysis of PCR products amplified from fungal DNA are each quite laborious.
Nucleic acid probes specific for particular Candida species have been developed (e.g., Elie et al., J. Clin. Microbiol., 36:3260-32655, 1998). Such probes have facilitated the development of methods for the rapid detection and identification of Candida species. For example, universal fungal primers can be used to amplify multicopy gene targets (such as the ITS2 region of the ribosomal DNA (rDNA) repeat region) from genomic DNA of Candida sp. present in a sample; then, amplicons can be detected using species-specific probes (e.g., Fujita et al., J. Clin. Microbiol., 33:962-967, 1995; Shin et al., J. Clin. Microbiol., 35:1454-1459, 1997; Shin et al., J. Clin. Microbiol., 37:165-170, 1999). DNA probes have been designed to specifically detect a variety of Candida species, including C. albicans, C. glabrata, C. guilliermondii, C. kefyr, C. krusei, C. lambica, C. lusitaniae, C. parapsilosis, C. pelliculosa, C. rugosa, C. tropicalis, C. zeylanoides, C. haemulonii, C. norvegensis, C. norvegica, C. utilis, C. viswanathii, and C. dubliniensis (e.g., Elie et al., J. Clin. Microbiol., 36:3260-32655, 1998).
Various methods of detecting the specific binding of Candida-specific probes to amplified target DNAs also have developed, including enzyme immunoassay (Elie et al., J. Clin. Microbiol., 36:3260-32655, 1998), and exonuclease cleavage of fluorescent reporter dyes from labeled-probes (Shin et al., J. Clin. Microbiol., 37:165-170). New technologies that can simultaneously detect large numbers of target nucleic acid sequences in a mixed sample (such as the multi-analyte profiling (MAP) system) have been and continue to be developed. Certain of such technologies can facilitate, for example, the high-throughput detection and classification of fungal pathogens in samples that may contain one or more such fungi. To implement this (and other more traditional) technologies, fungus-specific probes that distinguish among closely related fungi (such as species of Candida) are needed.